SGK1/FOXO3 Signaling in Hypothalamic POMC Neurons Mediates Glucocorticoid-Increased Adiposity

Despite the overwhelming beneficial anti-inflammatory effects of glucocorticoid, chronic glucocorticoid treatment has been shown to cause numerous adverse metabolic outcomes, including fat mass gain (1). Recent studies have elucidated several peripheral mechanisms underlying glucocorticoid-induced increase in fat mass. For example, glucocorticoid induces adipocyte differentiation (1–3), alters lipid metabolism in adipose tissue (1–3), and inhibits browning of white adipose tissue (WAT) or thermogenesis of brown adipose tissue (BAT) (4,5). In fact, body fat mass is largely controlled by the central nervous system (CNS) (6–8). Specific populations of neurons in the arcuate nucleus (ARC) of the hypothalamus also play fundamental roles in the regulation of energy balance and lipid metabolism (6–8). In particular, neurons coexpressing the orexigenic neuropeptides agouti-related protein and neuropeptide Y, and neurons coexpressing the anorexigenic proopiomelanocortin (POMC) precursor and the cocaine- and amphetamine-related transcript, are extensively involved in the regulation of appetite, body weight, and metabolism (6–8). POMC is a protein expressed and secreted from POMC neurons and cleaved by prohormone convertases to produce α-melanocyte-stimulating hormone (α-MSH) (8). α-MSH binds to the melanocortin 4 receptor and functions as a key hub linking the CNS to peripheral organs through the sympathetic nervous system (SNS), whereas dysfunction of this signaling axis leads to obesity in mice and humans (9,10). Activation of the SNS promotes the release of norepinephrine (NE) that binds to β-adrenergic receptor 3 and stimulates WAT lipolysis and BAT thermogenesis (11–14). Although previous studies have shown that glucocorticoid regulates food intake and energy expenditure (15,16), the central signals mediating the effect of glucocorticoid are poorly understood.

Serum- and glucocorticoid-regulated kinase 1 (SGK1) belongs to the family of serine/threonine kinases, and its coding region was originally isolated from rat mammary tumor cells (17). SGK1 is ubiquitously expressed in various tissues, including hypothalamus (17), and functions via activation of the glucocorticoid receptor (GR), retinoid X receptor, peroxisome proliferator–activated receptor γ, and nuclear factor κB (17). SGK1 is involved in the regulation of many processes, including hypertension, epithelial sodium channel activity, and insulin sensitivity (17–19). SGK1 also mediates many important functions of glucocorticoids, including insulin secretion and hippocampal neurogenesis (20,21). Although extensive studies have been carried out, a role for hypothalamic SGK1 in the regulation of energy homeostasis is unknown. Furthermore, SGK1 is well known as an early-response gene that can be induced by acute glucocorticoid treatment in various cells and animal models (20–22); however, the effect of chronic glucocorticoid treatment on SGK1 expression remains largely unknown. In fact, the expression of SGK1 in the context of glucocorticoid-induced metabolic effects could be very important, as studies show that sometimes SGK1 may have effects opposing those of glucocorticoid (23).

Despite the aforementioned unknown facts, we can speculate that SGK1, as a downstream target of glucocorticoid (17) expressed in the hypothalamus (17), may contribute to the central action of glucocorticoid. Therefore, the aim of this study was to test this hypothesis first by determining the expression of SGK1 in the hypothalamus and then by investigating its possible contribution to glucocorticoid-increased adiposity.

By creating mice that develop or experience adult-onset knockout of SGK1, or overexpression of SGK1 in POMC neurons, we demonstrate a crucial role for SGK1 expressed in POMC neurons in glucocorticoid-increased adiposity and provide a novel mechanistic link between glucocorticoid treatment and body fat mass gain.

POMC-Cre (24) and tamoxifen-inducible POMC-cre (POMC-cre:ER^T2) (24) mice were kindly provided by Joel K. Elmquist and Tiemin Liu from University of Texas Southwestern Medical Center (Dallas, TX); floxed SGK1 allele (SGK1^loxp/loxp) mice (18) were provided by Dr. Geza Fejes-Toth and Dr. Aniko Naray-Fejes-Toth (Geisel School of Medicine, Dartmouth College, Hanover, NH). To generate POMC neuron–specific SGK1 knockout (PSKO) mice, POMC-Cre mice were crossed with SGK1^loxp/loxp mice. To generate inducible POMC-specific SGK1 knockout mice, POMC-cre:ER^T2 mice were crossed with SGK1^loxp/loxp mice. Tamoxifen (0.15 g/kg; Sigma-Aldrich, St. Louis, MO) suspended in corn oil (Sigma-Aldrich) was intraperitoneally injected into 8-week-old male SGK1^loxp/loxp or SGK1^loxp/loxp × POMC-cre:ER^T2 littermate mice for five consecutive days to generate mice with adult-onset SGK1 deletion in POMC neurons (PSKO-ER). Dexamethasone (Dex) treatment was administered to male wild-type (WT), control, and PSKO mice, and to male POMC-Cre mice injected with adeno-associated virus (AAV) expressing constitutively active mutant rat SGK1 (S422D) (AAV-CA SGK1)/AAV-null in ARC (control/POMC neuron–specific SGK1 overexpression [PSOE] mice) by intraperitoneal injection of PBS or 5 mg/kg Dex every other day for 6 weeks or for 2 h (1,25). All the mice were on a C57BL/6J background. Mice were maintained under a 12-h light/12-h dark cycle (lights on at 0700 h/lights off at 1900 h) at 25°C, with free access to water and standard chow diet. In vivo studies were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Shanghai Institute for Nutritional Sciences, Chinese Academy of Sciences.

Intracerebroventricular (ICV) cannulation experiments were conducted as previously described (26). After surgery, the mice were given 5 days to recover and were then infused with 2 μL α-MSH peptide (Abcam, Cambridge, U.K.) at a concentration of 1 nmol/μL or 2 μL artificial cerebrospinal fluid (Tocris, Bristol, U.K.), and experiments were conducted 24 h later. ARC administration experiments were conducted as previously described (6). Mice were anesthetized and received bilateral stereotaxic injections of adenovirus expressing FOXO3-specific short hairpin RNA against FOXO3 (Ad-shFOXO3) or scrambled control adenovirus (Ad-scrambled), AAV-CA SGK1, or control AAV-null into the ARC (1.5 mm posterior to the bregma, ±0.2 mm lateral to the midline, and 6 mm below the surface of the skull). The AAV-CA SGK1 expression plasmid was constructed in pAAV-Ef1a-DIO-mCherry-2A plasmid (Addgene, Cambridge, MA), and SGK1 started to express only in the presence of CRE recombinase.

The body composition of mice was measured with a nuclear magnetic resonance system (Bruker, Rheinstetten, Germany). Indirect calorimetry was performed in a comprehensive laboratory animal-monitoring system (Columbus Instruments, Columbus, OH), as previously described (27). Rectal temperature of mice was measured at 1400 and 1700 h with a rectal probe attached to a digital thermometer (Physitemp Instruments Inc., Clifton, NJ). Food intake was measured as reported previously (6).

AI9 (tdTomato) reporter mice (The Jackson Laboratory) were mated with, or AAV-CA SGK1 and AAV-null expressed mCherry red fluorescent protein were injected into the ARC of, POMC-Cre mice to reflect POMC neurons, demonstrated by co-localization with POMC antibodies. The distribution and number of POMC neurons were determined as described previously (6). Average somatic area was analyzed in >500 POMC neurons (n = 4 mice/genotype). The area occupied by POMC neurons was manually scored using ImageJ software.

Hypothalamus was prepared as previously described (6), and α-MSH was quantified with an ELISA kit (Phoenix Pharmaceuticals Inc., Burlingame, CA), according to the manufacturer’s instructions.

Hypothalamic nuclear and cytoplasmic fractions were isolated as previously described (28).

Immunofluorescence staining was performed, as previously described (29), with anti-SGK1 and the anti-p-N-myc downstream-regulated gene 1 (p-NDRG1) (Abcam), anti-POMC (Phoenix Pharmaceuticals Inc.), anti-FOXO3 (Cell Signaling Technology, Danvers, MA), anti-p-SGK1 and anti-GR (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-α-MSH (Merck Millipore, Frankfurter, Germany). Immunofluorescence staining of p-FOXO3 was performed using the Tyramide Signal Amplification Cyanine 3 system (Perkin-Elmer, Boston, MA), and anti-p-FOXO3 primary antibody (Cell Signaling Technology) was coincubated with anti-mCherry (Abbkine, Inc., Wuhan, China).

RNA was isolated and RT-PCR performed as previously described (27). The sequences of primers used in this study are available upon request.

Western blotting was analyzed, as previously described (27), with the following primary antibodies: anti-p-FOXO3, anti-FOXO3, anti–lamin B1, and anti-p-GR (Cell Signaling Technology); anti-SGK1 and anti-GR (Abcam); anti–uncoupling protein 1 (UCP1) and anti-p-SGK1 (Santa Cruz Biotechnology); and anti-α-tubulin and anti-β-actin (Sigma-Aldrich).

Primary cultures of hypothalamic neurons were prepared as previously described (27). On day 7, primary cultured hypothalamic neurons were infected with adenovirus expressing SGK1-specific short hairpin RNA (10⁸ plaque-forming units/cells on 60 cm²) or Ad-scrambled, constructed as described previously (19). Primary hypothalamic neurons were transfected with small interfering RNA for FOXO3 using X-tremeGENE siRNA Transfection Reagent (Roche Diagnostics, Mannheim, Germany). Constitutively active mutant rat SGK1 (S422D) was subcloned into a PCMV-MYC plasmid and transfected into primary cultured hypothalamic neurons using Lipofectamine 2000 (Life Technologies).

All values are presented as the mean ± SEM. Differences between groups were analyzed by either the Student t test or one-way ANOVA followed by the Student-Newman-Keuls test. Differences for which P was <0.05 were considered statistically significant.

To investigate the metabolic effects of Dex, C57B6J WT mice were intraperitoneally injected with Dex for 6 weeks; this model has been commonly used to study the role of Dex (1,30). Dex treatment did not change body weight, although the total body fat and abdominal fat mass were increased compared with those in mice receiving the control treatment, possibly as a result of decreased lean mass (Supplementary Fig. 1A–D). A balance between energy intake and energy expenditure maintains body fat mass (7). Dex treatment did not change food intake but did decrease energy expenditure as measured by 24-h indirect calorimetry (Supplementary Fig. 1E and F). No difference was observed in locomotor activity, but body temperature, levels of the BAT thermogenic marker UCP1 (11), and levels of serum NE were significantly lower in Dex-treated mice (Supplementary Fig. 1G–J).

To investigate the possible involvement of hypothalamic SGK1 in Dex-increased adiposity, we examined hypothalamic SGK1 expression under this condition; it is interesting that we found that hypothalamic SGK1 and phosphorylated (p-)SGK1 were decreased in Dex-treated mice (Fig. 1A and B). Furthermore, immunofluorescence staining showed that SGK1 and p-SGK1 were decreased in the ARC of the hypothalamus of Dex-treated mice (Fig. 1C–F). Immunofluorescence staining of tdTomato and SGK1 showed that SGK1 was colocalized with POMC neurons in PBS-treated mice but was decreased significantly in POMC neurons of Dex-treated mice (Fig. 1G and H). By contrast, acute treatment increased SGK1 expression in the ARC of the hypothalamus (Fig. 1I–L).

Based on the above results, we speculated that knockout of SGK1 expression in POMC neurons might mimic Dex-induced metabolic alterations. To test this hypothesis, we generated PSKO mice. Immunofluorescence staining of tdTomato and SGK1 showed that SGK1 was colocalized with POMC neurons (>90% overlap with tdTomato) in control mice but was almost absent in POMC neurons of PSKO mice (Fig. 2A and Supplementary Fig. 2A–C), with no difference in SGK1 staining in the paraventricular nucleus (PVN) and ventromedial hypothalamus (VMH) between PSKO and control mice (Supplementary Fig. 2D–G). Sgk1 mRNA levels were consistently decreased ∼50% in the ARC, as the ARC of PSKO mice have other neurons or neurogliocytes (31,32), but Sgk1 mRNA levels were unchanged in other brain areas and tissues (Fig. 2B). Anatomical assessment of POMC neurons throughout the ARC area revealed no significant alterations in neuronal population size, distribution, or somatic area (Supplementary Fig. 3A and B), indicating that SGK1 deficiency did not alter POMC neuron differentiation and/or survival. Because the POMC promoter also drives CRE recombinase expression in the pituitary (33), we examined serum contents of hormones secreted from the pituitary, including corticosterone and growth hormone (34), and found that the levels of these two hormones were not altered in PSKO mice (Supplementary Fig. 3C and D).

Male PSKO mice exhibited significantly increased body weight from the age of 9 weeks compared with control mice (Fig. 2C); this was accompanied by a significant increase in total body fat and abdominal fat mass (Fig. 2D and E) but unchanged lean mass (Supplementary Fig. 2H). Food intake was not altered, but the energy expenditure was markedly decreased and the respiratory exchange ratio (RER; Vco₂/Vo₂) was higher in PSKO mice (Fig. 2F–H). Again, locomotor activity was not changed, but body temperature, UCP1 in BAT, and serum NE levels were significantly lower in PSKO mice (Fig. 2I–L). Female PSKO mice displayed phenotypes similar to those observed in male mice (Supplementary Fig. 4), so we performed all of the subsequent studies in male mice.

We next asked whether adult-onset loss of SGK1 in POMC neurons had effects similar to those of ablation during development. We used the POMC-cre:ER^T2 mouse model (24) that allows temporal control of CRE recombinase activity and can be combined with SGK1^flox/flox mice to produce mice with adult-onset deletion of SGK1 (PSKO-ER). We observed phenotypes similar to those found when using constitutive POMC-Cre mice (Supplementary Fig. 5).

We then asked whether overexpression of SGK1 in POMC neurons in mice would have the opposite phenotype of that observed in PSKO mice and whether Dex-increased adiposity would be avoided. For this purpose, we generated PSOE mice by bilateral stereotaxic injection into the ARC of AAV-CA SGK1 or control AAV-null male POMC-Cre mice. The effect of SGK1 overexpression was validated by immunofluorescence staining of the phosphorylated NDRG1, which reflects the activation status of SGK1 (35) (Fig. 3A), and increased SGK1 signals in POMC neurons (>90% overlap with mCherry), but not in the PVN and VMH, of PSOE mice (Supplementary Fig. 6A–H). As predicted, body weight decreased (starting from 6 weeks after AAV injection), accompanied by a decrease in total body fat and abdominal fat mass, in PSOE mice (Fig. 3B–D). Food intake was not affected, but the energy expenditure was increased and RER was decreased in PSOE mice (Fig. 3E–G). No difference was observed in locomotor activity, but body temperature, UCP1 in BAT, and serum NE levels were increased in PSOE mice (Fig. 3H–K). Furthermore, PSOE mice were resistant to Dex-induced fat accumulation and other metabolic alterations (Fig. 4), and Dex injected 5 weeks after AAV injection created no difference in lean mass and fat mass between control and PSOE mice (Supplementary Fig. 6I and J). By contrast, Dex had a very mild effect on PSKO mice, as demonstrated by the slightly decreased body weight and lean mass as well as increased fat mass, and no significant effect on food intake or energy expenditure (Supplementary Fig. 7).

Because previous studies have shown that α-MSH plays a critical role in the regulation of energy homeostasis (33), we asked whether it might be involved in Dex-induced metabolic alterations. As predicted, a dramatic reduction of α-MSH staining was observed in PVN of Dex-treated mice (Fig. 5A and B). Similar results were obtained in PSKO mice (Fig. 5C and D). The amount of α-MSH, as analyzed by ELISA, was consistently significantly decreased in the hypothalamus of PSKO mice (Fig. 5E). Notably, Dex-reduced α-MSH staining was reversed in PSOE mice (Fig. 5F and G).

To investigate whether α-MSH could mediate SGK1 regulation of energy homeostasis, we administered α-MSH peptide ICV to PSKO or control mice. ICV injection of α-MSH to PSKO mice markedly reduced body weight and abdominal fat mass and increased rectal temperature compared with those values in mice injected with the vehicle (control; Fig. 5H–J). ICV injection of α-MSH in PSKO mice also blocked a UCP1 protein decrease (Fig. 5K). Similar effects were observed in control mice after ICV injection of α-MSH (Fig. 5H–K).

α-MSH levels are determined by the levels of its precursor POMC and the expression of prohormone convertases that are responsible for cleaving POMC to α-MSH (8). The reduced α-MSH concentration in Dex-treated mice did not seem to be a consequence of decreased expression of processing enzymes, including prohormone convertase 1 (Pc1/3), prohormone convertase 2 (Pc2), carboxypeptidase E (Cpe), α-amidating monooxygenase (Pam), and prolylcarboxypeptidase (Prcp) (8), as gene expression of these enzymes was unchanged (Fig. 6A). On the other hand, POMC expression was decreased in Dex-treated mice (Fig. 6A–C). Similar results were obtained in PSKO mice (Supplementary Fig. 8A–C). The effect of Dex on reducing POMC expression, however, was reversed by overexpression of SGK1 (Fig. 6D and E). Similarly, SGK1 knockdown decreased Pomc expression and SGK1 overexpression increased Pomc expression in primary cultured hypothalamic neurons (Supplementary Fig. 8D and E).

We then investigated the downstream signaling of SGK1 in mediating Dex-decreased POMC expression. Previous studies showed that SGK1 phosphorylates FOXO3 (36) and that another member from the same FOXO family, FOXO1, inhibits Pomc expression (37), suggesting that FOXO3 might have a function similar to that of FOXO1 downstream of SGK1 in Dex-induced metabolic alterations. Consistent with this possibility, hypothalamic FOXO3 phosphorylation was decreased in Dex-treated mice (Fig. 6F). Similar reduction was observed in the hypothalamic ARC of PSKO mice (Supplementary Fig. 9A). Furthermore, Dex-decreased hypothalamic FOXO3 phosphorylation was reversed in PSOE mice (Fig. 6G and H). Similar regulatory effects of SGK1 on p-FOXO3 were observed in primary cultures of hypothalamic neurons (Supplementary Fig. 9B and C).

The inhibitory effect of SGK1 knockdown on Pomc expression was reversed by small interfering RNA–mediated FOXO3 inhibition (Supplementary Fig. 9D), thereby prompting us to investigate the in vivo function of FOXO3 downstream of SGK1. For this purpose, we knocked down FOXO3 expression in the ARC of PSKO and control mice by ARC administration (6) of adenovirus expressing short hairpin RNA directed against the coding region of FOXO3 (Ad-shFOXO3) (38) or Ad-scrambled. The effect of Ad-shFOXO3 was demonstrated through decreased expression of Foxo3 and the corresponding change in Pomc expression in the ARC of PSKO mice (Fig. 6I). Immunofluorescence consistently showed that FOXO3 was decreased in the ARC, but not the PVN or VMH, of these mice (Supplementary Fig. 10A and B). Ad-shFOXO3 decreased the body weight, total body fat, and abdominal fat mass in PSKO mice (Fig. 6J–L). Although food intake was not affected (Supplementary Fig. 10C), the decreased energy expenditure and increased RER in PSKO mice were largely reversed by Ad-shFOXO3 (Fig. 6M and N). No significant difference in locomotor activity was detected (Supplementary Fig. 10D); however, the decreased body temperature, UCP1 in BAT, and serum NE in PSKO mice were upregulated by Ad-shFOXO3 (Fig. 6O–Q). Moreover, the reduced α-MSH staining in PSKO mice was also blocked by Ad-shFOXO3 (Supplementary Fig. 10E and F). Except for the unaltered body weight, similar effects were observed in control mice following administration of Ad-shFOXO3 (Fig. 6I–Q and Supplementary Fig. 10).

Because glucocorticoid functions via the GR (28), we investigated the spatial regulation of GR and SGK1/FOXO3 using previously validated GR antibodies (39). Although hypothalamic Gr mRNA was unchanged, total GR and phosphorylated GR expression were significantly decreased in Dex-treated mice (Supplementary Fig. 11A and B). Furthermore, these three proteins were all expressed in POMC neurons and hypothalamic nuclear p-GR was decreased and FOXO3 was increased, but the cytoplasmic total and phosphorylated proteins examined were all decreased, in Dex-treated mice (Supplementary Fig. 11C and D).

Fat mass accumulation is a serious side effect of glucocorticoid therapy (1). Recent studies have elucidated several peripheral mechanisms underlying glucocorticoid-induced gains in fat mass (1–5). In this study we demonstrated a novel central mechanism, mediated by SGK1, underlying glucocorticoid-increased adiposity. SGK1 is a well-known downstream target of Dex (20–22). It has been widely demonstrated that acute Dex treatment induces SGK1 (20–22). It is interesting to note that we found decreased SGK1 expression in POMC neurons in the ARC of Dex-treated mice. The importance of POMC SGK1 in mediating Dex-induced adiposity was demonstrated by the observation that knockout of SGK1 expression in POMC neurons increased adiposity, whereas overexpression of SGK1 in POMC neurons resulted in a lean phenotype and prevented Dex-induced fat mass gain in mice. Furthermore, the Dex-induced gain in fat mass was much lower in PSKO than in control mice. Fat mass could, however, still be increased by Dex treatment in PSKO mice, suggesting the existence of other central or peripheral signals involved in Dex-increased adiposity (40). Our study provides a novel mechanistic link between glucocorticoid treatment and fat mass gain. This is important for understanding the mechanisms of glucocorticoid-induced metabolic phenotypes and provides an important hint for a possible treatment target for glucocorticoid-induced side effects. In addition, our study reports an unrecognized novel function of SGK1 in POMC neurons of the hypothalamus in the regulation of energy homeostasis. These results are important for understanding the signals in specific neurons that are critical for metabolic control.

A balance between energy intake and energy expenditure maintains body fat mass (7). BAT oxidizes fat to produce heat via increased expression of uncoupled proteins, which is stimulated by the activation of the SNS. Deletion of UCP1 induces obesity and upregulation of UCP1 increases thermogenesis and energy expenditure in mice (11). Other studies also showed that disruption of SNS activity has a significant negative impact on energy expenditure (6,41,42). Our study showed that Dex increased adiposity mainly by decreasing energy expenditure, as food intake was not changed in Dex-treated mice. Furthermore, the decrease in energy expenditure resulting from Dex treatment was most likely due to decreased thermogenesis in BAT, as demonstrated by the decreased body temperature, UCP1 expression in BAT, and serum NE in these mice. Lipolysis in WAT is also regulated by SNS activity (41,42), which might also affect Dex-induced adiposity and should be studied in the future.

Extensive evidence indicates that melanocortin signaling in the hypothalamus plays an important role in regulating energy homeostasis and lipid metabolism through its effects on SNS activity in BAT (11–14). In this study we demonstrated a possible role for α-MSH in mediating Dex regulation of adiposity, as α-MSH levels were decreased in Dex-treated mice via SGK1, and restoration of hypothalamic α-MSH levels by ICV administration of this peptide normalized inadequate energy homeostasis in PSKO mice. Although the beneficial effects of this pharmacological treatment are most likely mediated through direct actions on POMC neurons, we cannot exclude its potential effects on other hypothalamic areas as a result of the delivery route used.

Our results suggest that the reduced α-MSH content in Dex-treated mice was not caused by an altered proteolysis process, but the decreased Pomc expression is possibly due to glucocorticoid resistance, as Dex was shown to induce Pomc expression (43). Furthermore, we found that Dex decreased Pomc expression via the SGK1/FOXO3-dependent pathway, as the inhibitory effect of Dex on Pomc expression was blocked in mice with SGK1 overexpression or FOXO3 inhibition. Many studies, including those conducted on FOXO3 knockout mice, have demonstrated that FOXO3 is vital for many functions of the CNS and has roles in neural stem cell homeostasis, stress, and Huntington disease (44,45). We showed that it functions as a downstream signal of SGK1 in the regulation of energy homeostasis. We also demonstrated the spatial relationships among GR, SGK1, and FOXO3, providing a basis for the interaction among and regulation of these proteins.

In this study we also demonstrated that adult-onset loss of SGK1 in POMC neurons results in a phenotype similar to that of ablation during development. This is a key issue, because some works report that multiple hypothalamic neurons express POMC in adult mice (24) and prenatal and postnatal ablation of certain neurons result in disparate feeding behavior, suggesting that phenotypes caused by prenatal ablation may be influenced by developmental compensation (24). The POMC promoter also drives CRE recombinase expression in corticotrophs and melanotrophs (46). The contribution of the pituitary might not be that significant in this study, as no changes were observed in serum corticosterone and growth hormone, which reflect the function of the pituitary (34), between PSKO and control mice.

Previous studies have shown that POMC neurons are involved in the regulation of food intake (29,47). For reasons unknown, however, we found that food intake was not significantly affected by Dex treatment, or in PSKO or PSOE mice. Consistent with our study, however, previous works indicate that genetic blockade of the CNS–melanocortin 3 receptor promotes fat accumulation in the absence of hyperphagia (48).

In contrast to the stimulatory effect of glucocorticoid on SGK1 expression (20–22), we observed decreased hypothalamic SGK1 expression following chronic Dex treatment, which is, to our knowledge, a novel observation. We speculate that this inhibition is not a direct effect of Dex on SGK1 expression but rather a consequence of attenuated Dex-mediated signaling, as it has been previously shown that prolonged Dex treatment causes glucocorticoid resistance (49). Because glucocorticoid normally functions via the GR (28), the difference in SGK1 expression under acute or chronic Dex treatment may be caused by differences in GR activity under different conditions, as shown by our work and that of others (28,39). In addition, because chronic Dex treatment affects the activity of several regulatory molecules that influence SGK1 transcription and/or mRNA decay (28,50), and because hypothalamic signals might also be affected by peripheral events (6,16,26), the possible contribution of these factors to hypothalamic SGK1 expression cannot be excluded in Dex-treated mice. These possibilities will be explored in future studies.

In summary, our results demonstrate that SGK1/FOXO3 signaling in POMC neurons is crucial for Dex-induced adiposity. These results provide novel insights into the central mechanisms underlying Dex-induced obesity. In this study we also established that SGK1 in POMC neurons is an essential regulator of systemic energy balance. This previously unrecognized role for hypothalamic SGK1 also indicates a potential novel drug target in treating obesity and its related metabolic disorders.

Funding. This work was supported by grants from the National Natural Science Foundation of China (81325005, 81390350, 81471076, 81570777, 81130076, 31271269, 81400792, 81500622, and 81600623), a Basic Research Project of the Shanghai Science and Technology Commission (16JC1404900 and 17XD1404200), and the Chinese Academy of Sciences/State Administration of Foreign Experts Affairs International Partnership Program for Creative Research Teams. F.G. was supported by the One Hundred Talents Program of the Chinese Academy of Sciences.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. Y.D. researched data and wrote, reviewed, and edited the manuscript. Y.X., F.Y., Y.L., X.J., and J.D. researched data. G.F.-T. and A.N.-F.-T. generated and provided the floxed SGK1 mice. S.C. provided research material. Y.C., H.Y., and Q.Z. directed the project and contributed to the discussion. Y.S. and F.G. directed the project; contributed to the discussion; and wrote, reviewed, and edited the manuscript. Y.S. and F.G. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.